Warfarin (Coumadin), a vitamin K antagonist, is the most commonly prescribed oral anticoagulant. It is also notorious as one of the most unpredictable and dangerous drugs in current medical practice. The observed 20- fold range in optimal dose can be partially attributed to genetics and diet: for example, polymorphisms in genes linked to vitamin K recycling and warfarin metabolism, including CYP3A4, can change warfarin pharmacokinetics and function. Also, altered intake of leafy vegetables rich in phylloquinone, the plant-based form of vitamin K, can override the effects of the drug. Despite extensive studies with respect to these factors, nearly half of observed warfarin dose variation still remains unexplained. Notably, molecular characterization of vitamin K in the liver reveals that only 10% is the plant-based form: the remainder carries the chemical signature of bacterial biosynthesis. How the unique microbial communities (microbiota) that each of us harbors in the gastrointestinal tract influence variability in response to warfarin or other drugs is not understood. Solutions to these challenges could lead to safer and more effective warfarin therapy. Unlike polymorphisms in our human genomes, gut microbial communities can be rapidly modified and therefore both the drug and the patient (via their microbiota) could be attuned for maximum therapeutic benefit. This application presents a plan to identify the specific microbial mechanisms that determine warfarin response. I have established a system to measure warfarin function in gnotobiotic mice, whose microbiota can be precisely manipulated. I have discovered that mice lacking gut microbes exhibit profound hyper-responsiveness to warfarin, consistent with a role for microbiota-derived vitamin K in this process. However, I also discovered altered regulation of drug metabolizing enzymes (DMEs), including the murine homolog of human CYP3A4 (Cyp3a11). Colonization of germfree mice with complete gut microbial communities normalizes warfarin function and DME expression. In Aim 1, this system will be used to determine the impact of interpersonal variation in microbiota-derived vitamin K on warfarin function. In Aim 2, I will define how gut microbial regulation of host DME gene expression impacts warfarin metabolism and function. Successful completion of these aims will address critical questions to explain frequent and dangerous adverse reactions to a widely used drug. Such an understanding could open the door to new approaches for optimizing warfarin dosing or gut community composition for safer pharmacology. Further, these studies will highlight host-microbiota interactions that could influence the function of other vitamin-K dependent processes and drugs.